Cdc25 Inhibition and Cell Cycle Arrest by a Synthetic Thioalkyl Vitamin K Analogue1

[CANCER RESEARCH 60, 1317–1325, March 1, 2000] Cdc25 Inhibition and Cell Cycle Arrest by a Synthetic Thioalkyl Vitamin K Analogue1

Kenji Tamura, Eileen C. Southwick, Jeffrey Kerns, Katherine Rosi, Brian I. Carr, Craig Wilcox, and John S. Lazo2 Departments of Pharmacology [K. T., E. C. S., J. S. L.], Chemistry [J. K., K. R., C. W.], and Surgery [B. C.], University of Pittsburgh, Pittsburgh, Pennsylvania 15261, and Second Department of Internal Medicine [K. T.], Hiroshima University School of Medicine, Hiroshima 734, Japan

ABSTRACT suggesting that unlike vitamin K3, its inhibition is mediated by sulfhydryl arylation rather than oxidative stress (8). One proposed site for A synthetic vitamin K analogue, 2-(2-mercaptoethanol)-3-methyl-1,4- interaction is the catalytic cysteine(s) found in protein tyrosine phos- naphthoquinone or compound 5 (Cpd 5), was found previously to be a phatases that regulate cell proliferation (2). potent inhibitor of tumor cell growth. We now demonstrate that Cpd 5 Protein tyrosine phosphatases that have an essential role in cell arrested cell cycle progression at both G1 and G2-M. Because of the potential arylating activity of Cpd 5, it might inhibit Cdc25 phosphatases, cycle progression include the Cdc25 phosphatases, which activate which contain a cysteine in the catalytic site. To test this hypothesis, we Cdks. In mammalian cells, Cdc25 phosphatases are encoded by a examined the inhibitory activity of Cpd 5 against several cell cycle- multigene family consisting of Cdc25A, Cdc25B, and Cdc25C (9– relevant protein tyrosine phosphatases and found that Cpd 5 was a potent, 11). Each Cdc25 homologue controls distinct aspects of cell cycle selective, and partially competitive inhibitor of Cdc25 phosphatases. Fur- progression. Cdc25C dephosphorylates and activates the mitotic ki- thermore, Cpd 5 caused time-dependent, irreversible enzyme inhibition, nase Cdc2/cyclin B, which is required for entry into mitosis (12). consistent with arylation of the catalytic cysteine in Cdc25. Treatment of Cdc25A is important for entry into S-phase (13), whereas Cdc25B is cells with Cpd 5 blocked dephosphorylation of the Cdc25C substrate, essential for preinitiating G -M transition and S-phase progression Cdc2, and its kinase activity. Cpd 5 enhanced tyrosine phosphorylation of 2 (14). Cdc25A and Cdc25B have oncogenic properties in cells that both potent regulators of G1 transition, i.e., Cdk2 and Cdk4, and decreased the phosphorylation of Rb, an endogenous substrate for Cdk4 have mutated Ha-ras or loss of Rb1, the Rb susceptibility gene (15). kinase. Furthermore, close chemical analogues that lacked in vitro Cdc25 Cdc25A and Cdc25B are transcriptional targets of the c-myc oncogene inhibitory activity failed to block cell cycle progression and Cdc2 kinase (16) and are overexpressed in several tumor types and may reflect activity. Cpd 5 did not alter the levels of p53 or the endogenous cyclin- poor prognosis (15, 17–19). Unfortunately, potent and selective independent kinase inhibitors, p21 and p16. Our results support the hy- hibitors of Cdc25 phosphatases are currently unavailable but would be pothesis that the disruption in cell cycle transition caused by Cpd 5 was attractive candidates as potential anticancer agents. attributable to intracellular Cdc25 inhibition. This novel thioalkyl K In human hepatoma cells, vitamin K3 induces hyperphosphoryla- vitamin analogue could be useful for cell cycle control studies and may tion of p34cdc2 (Cdc2) kinase and decreases the protein tyrosine provide a valuable pharmacophore for the design of future therapeutics. phosphatase activity in cell lysates (20). Vitamin K3 and other naphthoquinone analogues inhibit Cdc25A in vitro, and one of these INTRODUCTION analogues has been shown to cause G1 arrest (21). The mechanism by The vitamin K family of molecules comprises the natural forms which the potent redox-deficient thioalkyl K vitamin analogue Cpd 5 inhibits cell growth is not known, although inhibition of Cdc25 has vitamin K1 (phylloquinone) and vitamin K2 (menaquinones) and the been hypothesized (2). Thus, we have examined the actions of Cpd 5 synthetic form vitamin K3 (menadione). These naphthoquinone-containing molecules inhibit tumor cell growth in culture, with vitamin and two other vitamin K analogues on protein tyrosine phosphatases, including Cdc25A, Cdc25B, and Cdc25C, as well as their antiprolif- K3 being more potent than either vitamin K1 or K2 (1). Vitamin K3 exhibits low toxicity to animals (2, 3) and can enhance the antipro- erative and cell cycle checkpoint activity. liferative effects of other clinically used anticancer agents (4), although it is toxic to humans (5). The growth-inhibitory actions of MATERIALS AND METHODS vitamin K3 have been ascribed to both sulfhydryl arylation and oxidative stress because of redox cycling (6, 7). We previously 3 Materials and Antibodies. tsFT210 cells were a generous gift from Dr. synthesized and characterized a thioalkyl K vitamin analogue, Cpd 5 Chris Norbury (Oxford University, Oxford, United Kingdom) and were main- (Fig. 1), with superior growth-inhibitory activity that also rapidly tained for no longer than 30 passages as described elsewhere (22). The enhances cellular protein tyrosine phosphorylation and causes apop- anti-Cdc2 (SC 54), anti-Cdk2 (SC 163G), anti-Cdk4 (SC 601G), anti-cyclin D1 tosis (8). The antiproliferative and antiphosphatase activity of Cpd 5 (SC 6281), anti-cyclin E (SC 481), anti-p53 (SC 1312), anti-p21 (SC 3976), is antagonized by exogenous thiols but not by nonthiol antioxidants, and anti-p16 (SC 1207) antibodies were purchased from Santa Cruz Biotech- nology (Santa Cruz, CA). Agarose conjugate of each antibody was used for immunoprecipitation. Anti-cyclin A antibody was purchased from Oncogene Received 8/26/99; accepted 1/6/00. The costs of publication of this article were defrayed in part by the payment of page Research Product (Cambridge, MA). Anti-phosphotyrosine antibody was pur- charges. This article must therefore be hereby marked advertisement in accordance with chased from Upstate Biotechnology (Lake Placid, NY). Phospho-Rb antibody 18 U.S.C. Section 1734 solely to indicate this fact. and Rb antibody were purchased from New England Biolabs, Inc. (Beverly, 1 Supported in part by Army Breast Grant DAMD17-97-1-7229, the Fiske Drug MA), and anti-GAPDH antibody was purchased from Chemicon International, Discovery Fund, and USPHS NIH Grants CA 78039 and CA 82723. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Inc. (Temecula, CA). Histone H1 was obtained from Boehringer Mannheim Biomedical Science Tower E-1340, University of Pittsburgh, Pittsburgh, PA 15261. Co. (Indianapolis, IN). [␥-32P]ATP (10 mCi/ml) was from Amersham Life Phone: (412) 648-9319; Fax: (412) 648-2229; E-mail: [email protected]. Science, Inc. (Arlington Heights, IL). 3 The abbreviations used are: Cpd 5, compound 5, 2-(2-mercaptoethanol)-3-methyl- Chemical Syntheses. To synthesize Cpd 5, we added 1,8-diaza- 1,4-naphthoquinone; Cpd 16, 2-methyl-3-(1-oxyoctyl)-1,4-naphthoquinone; Cpd 22, 2-hydroxy-3-methyl-1,4-naphthoquinone (phthiocol); THF, tetrahydrofuran; NMR, nu- bicyclo[5.4.0]un-dec-7-ene (0.07 ml, 0.7 mmol) dropwise to a solution of clear magnetic resonance; s, singlet; t, triplet; m, multiplet; brs, broad singlet; Cdk, menadione (5.154 g, 29.9 mmol) and 2-mercaptoethanol (2.10 ml, 29.9 mmol) cyclin-dependent kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MS, in 150 ml ether at room temperature. Stirring was maintained at room tem- mass spectrum; GST, glutathione S-transferase; OMFP, o-methyl fluorescein phosphate; perature for 23 h, and then 20 ml of 3.6 M HCl were added. The organic and ts, temperature sensitive; PTP1B, protein tyrosine phosphatase 1B; SC-␣␣␦9, 4-(benzyl- (2-[(2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylamino bu- aqueous phases were separated, and the aqueous layer was extracted with tyric acid; Rb, retinoblastoma; VHR, vaccinia H1-related phosphatase. ether. The combined organic layers were dried over magnesium sulfate, 1317

carbonate (2.40 g, 22.6 mmol) and 30% hydrogen peroxide (10 ml, 97.9 mmol) in water (50 ml) was added together to a solution of menadione (10.168 g, 59.1 mmol) in warm ethanol (110–135 ml). The yellow quinone color disappeared, and the flask was then cooled in ice. Water was added (300 ml) to give 16.334 g of a white solid; melting point 87°C. 1.076 g of this solid was dissolved in concentrated sulfuric acid (6 ml), giving a deep red solution. The flask was swirled intermittently for 10 min, and then water (20 ml) was added slowly to give a yellow precipitate. The mixture was filtered, and the filtercake was washed with water until the filtrate was no longer acidic. This procedure afforded 0.724 g (67%) of Cpd 22; melting point 170–171°C. 1H NMR ␦ 3 (CDCl3) 8.15–8.07 (m, 2H), 7.79–7.66 (m, 2H), 7.3 (s, 1H), 2.12 (s, [ H]); 13 C NMR (CDCl3) 185.08, 181.23, 153.21, 134.89, 132.96, 129.47, 126.78, 126.18, 120.60, 8.74; IR (KBr) cmϪ1 3312 (brs), 1643 (s), 1579 (m); UV (ethanol) ␭max (log ⑀) 206 (4.54), 242 (4.48), 252 (4.47), 276 (4.65); MS (m/z): 188 (100), 160 (30), 132 (42), 105 (33), 77 (35); high resolution MS:

calculated for C11H8O3: 188.0473442, found: 188.049347. To synthesize Cpd 16, we added a solution of Cpd 22 (0.713 g, 3.793 mmol) in dry THF (4 ml) via cannula to a suspension of potassium hydride (0.229 g, 5.711 mmol) in dry THF (10 ml) at 0°C. The resulting dark brown mixture was stirred for 5–10 min when a solution of 18-Crown-6 (1.542 g, 5.841 mmol) in dry THF (4 ml) was added. In some reactions, we used supplemental THF to Fig. 1. Chemical structures of vitamin K1 and several analogues. aid in the stirring of the solution. The resulting burgundy mixture was stirred for 20 min, and then 1-iodooctane (0.68 ml, 3.768 mmol) was added. The mixture was refluxed for 21 h and then stirred at room temperature for 24 h. filtered, and concentrated to give 8.223 g of a dark brown viscous liquid. The reaction was quenched with saturated ammonium chloride and extracted Purification by flash chromatography using 30% ethyl acetate/hexanes to elute with ether. The organic and aqueous phases were separated, and the aqueous the first two bands, followed by 50% ethyl acetate/hexanes, gave 2.698 g 1 ␦ layer was extracted with ether. The combined organics were dried over (36%) of an orange solid: melting point 78–80°C. H NMR (CDCl3) 8.08–8.03 (m, 2H), 7.71–7.68 (m, 2H), 3.80 (t, J ϭ 5.7, 2H), 3.34 (t, J ϭ 5.8, magnesium sulfate, filtered, and concentrated to give 1.704 g of a dark brown 13 liquid. Purification by flash chromatography using 5% ethyl acetate/hexanes 2H), 2.50 (s, 1H), 2.38 (s, 3H); C NMR (CDCl3) 182.10, 181.42, 147.85, 1 145.87, 133.74, 133.41, 132.61, 131.86, 128.78, 126.55, 62.02, 37.11, 15.40; gave 1.11 g (98%) of a yellow solid: melting point, 38°C. H NMR (CDCl3) IR (KBr) cmϪ1 3292 (m), 1657 (s), 1585 (s), 1554 (s); UV (ethanol) ␭max (log ␦ 7.85–7.80 (m, 2H), 7.53–7.46 (m, 2H), 4.22 (t, J ϭ 6.6, 2H), 1.94 (s, 3H), ϭ 13 ⑀) 204 (4.21), 260 (4.22), 408 (3.33); MS (m/z): 248 (2), 230 (63), 221 (100), 1.68–1.59 (m, 2H), 1.35–1.16 (m, 10H), 0.77 (t, J 6, 3H). C NMR (CDCl3)

197 (73); high resolution MS: calculated for C13H10O2S: 230.0412, found: 185.26, 180.90, 157.20, 133.33, 132.84, 131.70, 131.40, 131.22, 125.81, 73.51, 230.0405. 31.67, 30.42, 29.18, 29.12, 25.69, 22.52, 13.97, 9.15; IR (KBr) cmϪ1 1668 (s), To synthesize Cpd 22, we used Fieser’s method. A solution of sodium 1620 (s) 1593 (s); UV (ethanol) ␭max (log ⑀) 208 (4.20), 248 (4.29), 276

Fig. 2. Inhibition of tsFT210 cell cycle progression at G2-M by Cpd 5 tsFT210 cells cultured at the permissive temperature of 32.0°C (A) and then incubated for 17 h at the nonpermissive temperature of 39.4°C (B). Cells were released from cycle arrest by shifting to the 32.0°C medium. The cells were then incubated for6hinthepresence of DMSO vehicle (C), 1 ␮M nocodazole (D), 10 ␮M Cpd5(E), 20 ␮M Cpd5(F), 20 ␮M Cpd 16 (G), 20 ␮M Cpd 22 (H), or 100 ␮M SC-␣␣␦9(I). Vertical bars, fluorescence corresponding to 2C and 4C DNA content. Results are representative of at least three independent experiments.

1318

Fig. 3. Inhibition of tsFT210 cell cycle progression at G1 by Cpd 5 tsFT210 cells were cultured at the permissive temperature of 32.0°C (A) and then incubated for 17 h at the nonpermissive temperature of 39.4°C (B). Cells were released from the G2-M block by being incubated at 32.0°C for 6h(C) and then incubated for an additional6hinthe presence of various agents. These were: DMSO vehicle (D), 50 ␮M roscovitine (E), 5 ␮M Cpd5(F), 10 ␮M Cpd5(G), 15 ␮M Cpd5(H), 20 ␮M Cpd5(I), or 100 ␮M SC-␣␣␦9(J). Vertical bars, fluorescence corresponding to 2C and 4C DNA contents. Results are representative of at least three independent experiments.

(4.07), 334 (3.51); MS (m/z): 316 (15) 201 (30), 188 (100), 172 (45), 160 (30); Western Blotting and Immunoprecipitation Studies. tsFT210 cells were

high resolution MS: calculated for C19H24O3: 300.1725, found: 300.1745. harvested and sonicated in the lysis buffer using the same procedure for cell ϫ 5 Flow Cytometric Analysis. tsFT210 cells were plated at 2 10 cells/ml synchronizing and drug exposure as described above for the G1 flow cytomet- and maintained at 32.0°C as described previously (22). Cell proliferation was ric analysis. For the phospho-Rb study, we harvested the cells at each time

blocked at G2 phase by incubation at 39.4°C for 17 h. The synchronized cells were point: -6 h (releasing point from the G2-M synchronizing); and -3, 0, 1.5, 3, and then released by reincubating at 32.0°C and treated immediately with Cpd 5, Cpd 6 h after treatment with 20 ␮M Cpd 5. The protein lysates were analyzed by ␣␣␦ 16, Cpd 22, or SC- 9, respectively, to probe for G2-M arrest. Cells were treated Western Blot for phospho-Rb, Rb, GAPDH, p53, p21, and p16. Immunopre- 6 h after G2-M release to determine G1 arrest. For both G2-M and G1 blockage cipitation assays were performed essentially as described previously (24), studies, treated cells were incubated at 32.0°C for an additional 6 h after each drug except we replaced 0.1% Tween 20 for 1% Triton X-100 in the lysis buffer. exposure and then harvested with PBS at 5 ϫ 105 cells/ml. The harvest cells were We incubated 2 mg of protein lysate on a rocker platform with 10 ␮gof stained with a solution containing 50 ␮g/ml propidium iodide and 250 ␮g/ml anti-Cdk2 or anti-Cdk4 agarose conjugate for4hat4°C. The immunocom- RNase A. Flow cytometry analysis was conducted with a Becton Dickinson FACS plexes were washed four times with the same lysis buffer. After the final wash, Star (Franklin Lakes, NJ). Each compound was tested at least three independent the immunocomplexes were suspended with SDS-electrophoresis loading times. A final concentration of 0.5% DMSO was used for all compounds and as buffer and analyzed by Western blotting for Cdk2, tyrosine phosphorylated a negative control. For positive controls, we used 100 ␮M SC-␣␣␦9 (for both Cdk2, Cdk4, tyrosine phosphorylated Cdk4, cyclin A, cyclin E, and cyclin D1 ␮ ␮ G2-M and G1), 1 M nocodazole (for G2-M) ,or 50 M roscovitine (for G1). as described above. To quantify the phosphorylation level of Cdk2 or Cdk4, we Enzyme Assays. The preparation of plasmid DNA and GST-fusion pro- scanned X-ray films and analyzed band intensity on a Molecular Dynamics teins has been described previously (23). The activities of the GST-fusion personal SI densitometer and analyzed them using the Image Quant software

Cdc25A, Cdc25B2, Cdc25C, and VHR, as well as human recombinant PTP1B, package (Ver. 4.1; Molecular Dynamics, Sunnyvale, CA). The phosphoryla- were measured as described previously (23) in a 96-well microtiter plate using tion level (pCdk2/Cdk2) was calculated by using the formula; pCdk2/ the substrate OMFP (Molecular Probes, Inc., Eugene, OR), which is readily Cdk2 ϭ (a)/(b), where a was the intensity of the phosphorylated Cdk2 band metabolized to the fluorescent o-methyl fluorescein. OMFP concentrations and b was the intensity of the Cdk2 band, respectively. Statistical significance ␮ approximating the Km were used: Cdc25A, Cdc25B2 and Cdc25C, 40 M; was analyzed using Student’s unpaired t test. VHR, 10 ␮M; and PTP1B, 200 ␮M. Inhibitors were resuspended in DMSO, and Cdc2 Assays. tsFT210 cells were synchronized, exposed to drugs, and

all reactions including controls were performed at a final concentration of 7% harvested as described above for the G2-M flow cytometric analysis. The DMSO. The final incubation mixture (150 ␮l) was optimized for enzyme protein lysates were analyzed by Western blot for Cdc2 as described previ- activity and comprised 30 mM Tris (pH 8.5 for Cdc25 phosphatases; pH 7.5 for ously (25). Cdc2 kinase activity assay was performed as described previously VHR and PTP1B), 75 mM NaCl, 1 mM EDTA, 0.033% BSA, and 1 mM DTT. (26). Briefly, the Cdc2 immunoprecipitates were incubated in 20 ␮l of kinase Reactions were initiated by adding 1 ␮g of Cdc25 phosphatases, 0.025 ␮gof reaction buffer (26) for 30 min at 37°C, with 3 ␮g of histone H1, 20 mM ␮ ␮ ␮ ␥ 32 VHR, or 0.25 g of PTP1B phosphatase. Fluorescence emission from the Tris-HCl, 10 mM MgCl2,5 M cold ATP, and 10 Ci of [ - P]ATP. The product was measured over a 20–60 min reaction period at ambient temper- proteins were separated by SDS-PAGE and analyzed with a Molecular Dy- ature with a multiwell plate reader (PerSeptive Biosystems Cytofluor II; namics STORM 860 PhosphorImager (Sunnyvale, CA). Framingham, MA; excitation filter, 485/20; emission filter, 530/30). For all enzymes, the reaction was linear over the time used in the experiments and was RESULTS directly proportional to both the enzyme and substrate concentration. Best Cpd 5 Arrested Synchronous tsFT210 Cell Cycle Progression at curve fit for Lineweaver-Burk plots and Kis was determined by using the curve-fitting programs Prism 3.0 (GraphPad Software, Inc., San Diego, CA) G2-M. We initially determined whether Cpd 5 blocked cell cycle and EZ-Fit 5.03 (Perrella Scientific, Inc., Amherst, NH). progression through checkpoints using murine tsFT210 cells, because 1319

2D). To determine the effect of Cpd 5 on G2-M cell cycle transition, we treated cells with either 10 or 20 ␮M Cpd 5 for 6 h after releasing cells at 32.0°C. As indicated in Fig. 2, E and F, both concentrations

of Cpd 5 significantly arrested cells at G2-M phase. This G2-M inhibition was selective to Cpd 5 and not seen with two structural

analogues, i.e., Cpd 16 and Cpd 22 (Fig. 1, G and H). The G2-M inhibition was similar to that seen with another inhibitor of the Cdc25 family of phosphatases, SC-␣␣␦9, which is structurally unrelated (Refs. 23 and 27; Fig. 2I). Cpd 5 Arrested Synchronous tsFT210 Cell Cycle Progression at

G1. We next examined whether Cpd 5 caused G1 arrest in tsFT210 cells. To investigate the mechanism of G1 cell cycle block by Cpd 5, we arrested tsFT210 cells at G2-M by shifting to the nonpermissive temperature, then released them into G1 by shifting to the permissive temperature, and subsequently added either Cpd 5 or DMSO vehicle 6 h later. Cells that were treated with the DMSO vehicle passed

through G1 phase as expected and produced the broad S-phase peak (Fig. 3D), whereas cells exposed continuously to 50 ␮M roscovitine

were blocked and did not pass through G1 (Fig. 3E). As illustrated in Fig. 3, F–I, cells treated with 5 or 10 ␮M Cpd 5 were delayed, whereas ␮ cells treated with 15 or 20 M Cpd 5 were fully blocked at G1.In ␮ contrast, neither Cpd 16 nor Cpd 22 at 20 M blocked G1 transition (data not shown). As expected from our previous studies (Fig. 2), Cpd

5 not only caused a G1 block but also prevented cells that were in the G2 phase from progressing through G2-M, which resulted in two prominent cell cycle peaks (Fig. 3, H and I). This dual G1 and G2-M inhibition was similar to that seen with a much higher concentration

Fig. 4. Inhibition of recombinant human phosphatases by vitamin K analogues. A, human recombinant Cdc25B2, VHR, or PTP1B was incubated with each vitamin K3 analogue (30 ␮M) at room temperature for 0–60 min, and inhibition was determined as f u Ⅺ ϭ described in “Materials and Methods.” , Cdc25B2; , VHR; , PTP1B (n 3). Bars, ␮ SE. B, Cdc25B2 was incubated at room temperature with each compound at 0.1–100 M for 0–60 min. The percentage of inhibition by Cpd 5 (f), Cpd 16 (‚), or Cpd 22 (E; n ϭ 3) is shown. C, selectivity of inhibition. The concentration-dependent inhibition f F ࡗ profile for inhibition of GST fusion proteins Cdc25B2 ( ), VHR ( ), and PTP1B ( )is shown. Activities of GST fusion phosphatases were assayed as described in “Materials and Methods.” Each value is the mean of three independent experiments.

Fig. 5. Time- and concentration-dependent phosphatase inhibition by Cpd 5. A, time-dependent inhibition of GST-Cdc25B2. The enzymes were either preincubated at they can be readily synchronized with exogenous compounds because room temperature with either 0.5% DMSO or 2 ␮M Cpd 5 for 0, 10, 20, 30, 60, or 90 min. of a ts Cdc2 (22). When tsFT210 cells were incubated at the permis- The reaction was initiated by addition of substrate OMFP. Activities of GST fusion phosphatases were assayed as described in “Materials and Methods.” The percentage of sive temperature of 32.0°C, they had a normal cell cycle distribution inhibition was determined by comparison to the DMSO control at each time point. Each (Figs. 2A and 3A); when cells were incubated at the nonpermissive value is the mean of three independent experiments and the SEs are indicated by bars unless they are less than the symbol size. B, irreversibility of Cdc25B2 inhibition. temperature of 39.4°C, they arrested at G2-M, because of Cdc2 ␮ GST-Cdc25B2 was incubated with 2 M Cpd 5 at room temperature for 30 min. The inactivation (Ref. 22; Figs. 2B and 3B). When G2-M arrested cells reaction mixture was centrifuged in a Centricon 30 concentrator (Amicon, Inc., Bedford, were cultured at the permissive temperature for 6 h with DMSO MA), then washed three times with assay buffer to remove Cpd 5 from the enzyme. At time points 0, 15, 30, 45, 60, and 90 min after Cpd 5 removal, the enzyme solution was vehicle alone, we saw clear evidence of entry into G1 (Fig. 2C). In assayed for phosphatase activity by the addition of substrate OMFP, as described in ␮ contrast, 1 M nocodazole blocked cell passage through G2-M (Fig. “Materials and Methods.” 1320

22. The selectivity of Cpd 5 is illustrated in Fig. 4C; the IC50s for VHR and PTP1B were 45 and 3200 ␮M, respectively. We also found that 40 and 80 ␮M Cpd 5 lacked any significant inhibitory activity against recombinant human mitogen-activated protein kinase (data not shown).

The inhibition of Cdc25B2 was dependent on the length of enzyme exposure to Cpd 5; a 30-min preincubation with 2 ␮M Cpd 5 caused almost 50% more inhibition in enzyme activity than in samples that were exposed to Cpd 5 at the time of substrate addition (Fig. 5A). Preincubation longer than 30 min did not produce greater inhibition, possibly because Cpd 5 became inactivated. No reduction in enzyme

activity was seen when Cdc25B2 was preincubated with 0.5% DMSO for 90 min or less. The time-dependent inhibition was irreversible; a 90-min incubation in Cpd 5-free buffer did not restore the lost enzyme activity (Fig. 5B). Similar results were seen with both Cdc25A and VHR (data not shown). A kinetic analysis of the inhibition indicated a partial competitive inhibition for full-length human Cdc25A,

Cdc25B2, and Cdc25C (Fig. 6). The Kis for Cdc25A, Cdc25B2, and Cdc25C were 15, 1.7, and 1.3 ␮M, respectively. Cpd 5 Increased the Phosphorylation Level of Cdc2 in Syn- chronous tsFT210 Cells. One of the putative, endogenous, cellular substrates for both Cdc25B and Cdc25C is the mitotic inhibitor Cdc2, which must be dephosphorylated to allow entry into mitosis (14, 22, 28). Thus, we reasoned that an effective Cdc25 inhibitor would not

only cause a G2-M cell cycle block but would also prevent Cdc2 dephosphorylation. We, therefore, performed Western blotting on tsFT210 cell extracts to determine the Cdc2 phosphorylation levels in the presence or absence of Cpd 5. Protein lysates of tsFT210 cells

arrested at the G2-M boundary were harvested and analyzed by SDS-PAGE. Approximately 50% of Cdc2 was in the mitotic-inactive, hyperphosphorylated form, as reflected by a slower migrating Cdc2 (Fig. 7A). The phosphorylation of Cdc2 decreased gradually after cells

were released from G2-M block, and most of the Cdc2 was dephosphorylated 6 h after G2-M release, even in the presence of the DMSO vehicle (Fig. 7A). When we incubated cells with 1 ␮M nocodazole,

which caused a G2-M arrest, no hyperphosphorylation of Cdc2 was seen (Fig. 7B), consistent with its proposed inhibitory activity after Cdc2 activation. In contrast, Cdc2 dephosphorylation was partially blocked (ϳ70%) with 10 ␮M Cpd 5 and completely blocked (94%) with 20 ␮M (Fig. 7C). The Cdc2 phosphorylation status after 6 h with DMSO alone was similar in Fig. 7, A and C. SC-␣␣␦9at50␮M also Fig. 6. Kinetic analyses of Cdc25A, Cdc25B2, and Cdc25C inhibition by Cpd 5. Inhibitor concentrations: Œ,0␮M; , 0.3 ␮M; ࡗ,1␮M; F,3␮M; Ⅺ,10␮M; f,30␮M. caused hyperphosphorylation of Cdc2 (Fig. 7B). Because the phos- Double-reciprocal plots of inhibition by Cpd 5 of Cdc25A (A), Cdc25B2 (B), and Cdc25C phorylation status of Cdc2 determines its enzymatic activity (29), we (C) are shown. Enzyme activities were determined as outlined in “Materials and Meth- ods.” Best curve fit for Lineweaver-Burk plots and Kis were determined by using the examined the kinase activity of immunoprecipitated Cdc2 by meas- curve-fitting programs Prism 3.0 (GraphPad Software, Inc.) and EZ-Fit 5.03 (Perrella uring histone H1 phosphorylation in vitro. We found that the Cdc2 Scientific, Inc.). kinase activity in cells treated with 1 ␮M nocodazole was significantly increased, which was consistent with a previous study using tsFT210 of the structurally unrelated and less potent Cdc25 inhibitor, SC-␣␣␦9 cells (26). The Cdc2 kinase activity in cells treated with 10–20 ␮M (Fig. 3J). Cpd 5 was markedly reduced (Fig. 7D). The congeners, Cpd 16 and

Cpd 5 Is a Selective Inhibitor of Cdc25. Because of the dual G1 Cpd 22, however, did not block this kinase activity as expected by and G2-M blockage with Cpd 5 and previous speculation concerning their lack of effect on Cdc25 activity (Fig. 4A). Similar amounts of possible phosphatase inhibitory activity (8), we examined the inhib- Cdc2 were immunoprecipitated in cells treated with Cpd 5 (Fig. 7E). itory activity of Cpd 5, Cpd 16, and Cpd 22 (Fig. 1) against the dual Cpd 5 Increased Cdk2 and Cdk4 Tyrosine Phosphorylation in specificity phosphatases Cdc25B2 and VHR and the tyrosine phos- Synchronous tsFT210 Cells. Cdk4 plays a central role in regulating ␮ Ͼ phatase PTP1B. At 30 M, Cpd 5 caused 75% inhibition of recom- the G1 transition by its association with cyclin D1 (30). This complex binant human Cdc25B2 activity with only a small effect on VHR and remains inactive until Cdc25A dephosphorylates it. Cdk2 is also no inhibition of PTP1B (Fig. 4A). In contrast, identical concentrations involved in regulating the G1-S transition by its association with of the close structural analogues, Cpd 16 and Cpd 22, did not inhibit cyclin E or cyclin A. The Cdk2/cyclin E complex has been shown to any of these protein phosphatases, indicating the essential nature of be dephosphorylated at Thr-14 and Tyr-15 and, thereby, activated by ␤ the -mercaptoethanol moiety for enzyme inhibition. A more exten- Cdc25A treatment in vitro (31). To clarify the mechanism of G1 cell Ϯ ␮ Յ ␮ sive study revealed that the Cdc25B2 IC50 for Cpd 5 was 3.8 0.6 M cycle block by Cpd 5, we treated tsFT210 cells with 20 M Cpd 5 compared with Ͼ150 ␮M for the close analogues Cpd 16 and Cpd 22 for 6 h, immunoprecipitated Cdk2 or Cdk4 from the cell lysates, and (Fig. 4B). Thus, Cpd 5 was 40-fold more active than Cpd 16 or Cpd then determined tyrosine phosphorylation by Western blotting, using 1321

Fig. 7. Inhibition of Cdc2 dephosphorylation and kinase activity by Cpd 5 in synchronous tsFT210 cells. G2-M synchronous tsFT210 cells were treated with vehicle or various compounds and permitted to reenter the cell cycle by culturing at 32.0°C. We isolated protein lysates from cells that were not incubated (0h) or from cells incubated for6hatthe permissive temperature in the presence of a compound or vehicle. The protein lysate were analyzed by Western blotting for Cdc2 or Cdc2 kinase activity assay as described in “Materials and Methods.” A, DMSO control. B, nocodazole (1 ␮M) and SC-␣␣␦9 (50 ␮M)at6h.C, Cpd 5 or DMSO control at 6 h. D, Cdc2 kinase activity with histone H1 as a substrate. E, Cdc2 content in immunoprecipitates from Cpd 5-treated cells. P, phosphorylated; U, unphosphorylated.

anti-phosphotyrosine monoclonal antibody. As illustrated in Fig. 8, ation increased with passage into G1 phase (Fig. 10). Within 1.5 h the phosphorylation of both Cdk2 and Cdk4 increased after Cpd 5 after exposure of cells to Cpd 5, however, there was a marked treatment. We confirmed that there was equivalent loading of Cdk2 or inhibition of Rb phosphorylation with no alteration of Rb protein Cdk4 with anti-Cdk2 or anti-Cdk4 antibody, respectively (Fig. 8). To levels. Equivalent loading was confirmed by measuring GAPDH (Fig. quantify the phosphorylation level of Cdk2 or Cdk4, we determined 10). Thus, our results support the hypothesis that Cpd 5 blocked cell the intensity of the bands by densitometer and calculated a phospho- cycle progression through the G1 checkpoint by disruption of func- rylation level as described in “Materials and Methods.” Both Cdk2 tional Cdk activity through inhibition of Cdc25A activity. and Cdk4 tyrosine phosphorylation increased in a concentration- Cpd 5 Did Not Alter p53, p21, or p16 Levels. To ensure that dependent manner after Cpd 5 treatment, with a Ͼ5-fold increase the inhibition of Cdk4 kinase activity and cell cycle arrest were not being seen after exposure to 20 ␮M Cpd 5. secondary to p53 induction or increased Cdk inhibitors, we meas- Cpd 5 Does Not Affect Cyclin Interactions with Cdk2 or Cdk4. ured p53, p21, and p16 levels in tsFT210 cells after Cpd 5 treat- Cdk requires noncovalent interactions with cyclins to be functional. ment (Fig. 11). tsFT210 cells, which had been treated with an To exclude the possibility that Cpd 5 simply blocked such an intra- equitoxic etoposide concentration, displayed elevated p53 levels, cellular interaction, we treated tsFT210 cells with Cpd 5 for 6 h, whereas Cpd 5 produced no increase (Fig. 11A). We also saw no immunoprecipitated Cdk2 from cell lysates with an anti-Cdk2 anti- increase in p21 or p16 with Cpd 5 (Fig. 11, B and C), suggesting body, and then examined the immunoprecipitate for cyclin A and E that the dual cell cycle phase arrest was not due simply to non- content by Western blotting. We also immunoprecipitated Cdk4 from specific cell stress or DNA damage. cell lysates with anti-Cdk4 and determined cyclin D1 protein levels. Cyclin A or E association with Cdk2 was unchanged after Cpd 5 DISCUSSION treatment (Fig. 9A). Similarly, Cdk4 association with cyclin D1 was unaffected by the Cpd 5 treatment (Fig. 9B). For both analyses, we The Cdc25 dual-specific phosphatases have an essential role in loaded equivalent amounts of Cdk2 or Cdk4 as detected with anti- controlling cell proliferation by regulating the activities of Cdks (14,

Cdk2 or anti-Cdk4, respectively (Fig. 9). 31). In higher eukaryotes, Cdc25A is responsible for governing G1 Cpd 5 Decreases the Phosphorylation of Rb. The phosphoryla- transition into S phase, Cdc25B probably initiates cell cycle move- tion of Rb, which is a critical regulator of the G1 checkpoint, is ment through the G2 phase, and Cdc25C is required for entry into controlled in part by Cdk2. Thus, we examined the phosphorylation mitosis, because of its ability to dephosphorylate and activate Cdc2. status of Rb in synchronous tsFT210 cells at various times after Because Cdc25A and Cdc25B have also been reported to be onco- addition of 20 ␮M Cpd 5 (Fig. 10). As expected, the Rb phosphoryl- genic (15) and to be overexpressed in several tumor types (17, 18), 1322

Fig. 8. Effect of Cpd 5 on Cdk4 or Cdk2 tyrosine phosphorylation in tsFT210 cells. Synchronized tsFT210 cells were cultured for6hat32.0°C and then incubated for an additional6hinthepresence of 0 ␮M (Lane 1), 10 ␮M (Lane 2), or 20 ␮M (Lane 3) of Cpd 5. The cells were harvested and sonicated in lysis buffer and probed for tyrosine phosphorylation and total Cdk by Western blot as described in “Materials and Methods.” A, Cdk2 protein content and phosphorylation. B, Cdk4 protein content and ,ءءء ;P Ͻ 0.01 ,ءء ;P Ͻ 0.05 ,ء .phosphorylation P Ͻ 0.005. N.S., not significant. Bars, SE.

Cdc25 is an attractive therapeutic target. Although the Cdc25 family Previously, we reported that the thioether vitamin K analogue Cpd members appear to have distinct biological functions and possibly 5 was a more potent inhibitor of hepatoma cell proliferation than other substrates, the amino acids comprising their active site HC(X5)R K vitamins (8). Hepatoma cells normally only arrest in G1. Moreover, region are identical, suggesting that inhibitors with specificity to all we found that growth-inhibitory concentrations of Cpd 5 caused a three Cdc25s are feasible. Moreover, significant structural differences rapid increase in protein tyrosine phosphorylation that could be exist among the other protein tyrosine phosphatases and Cdc25 (32). blocked by elevating intracellular stores of thiols, such as cysteine Thus, it may be possible to identify selective inhibitors of this family (36, 39). Although we proposed sulfhydryl arylation of protein phos- of enzymes. phatases as a potential mechanism for enhanced phosphorylation and Except for the widely used broad-spectrum protein phosphatase growth inhibition, no experimental examination of the effects of Cpd inhibitor vanadate (33), few dual-specificity protein phosphatase in- 5 on specific phosphatases was performed previously. We now report hibitors have been reported (34, 35). Moreover, these analogues are that Cpd 5 inhibited Cdc25 in a partially competitive manner that was generally in limited supply, and the effects of these compounds on cell time dependent and ultimately irreversible. The Cdc25 enzymes share cycle transition or other enzymes are not known. We have previously a conserved COOH-terminal catalytic domain containing the Cys- ␣␣␦ synthesized and evaluated a small molecule, SC- 9, that was (X)5-Arg motif. In Cdc25A and presumably other Cdc25 enzymes, among the most potent of the known synthetic inhibitors of the Cdc25 Cys-430 forms a disulfide bond with Cys-384 that may be self- dual-specificity phosphatases (23). As noted in our current studies and inhibiting and redox sensitive (32). In contrast to other K vitamins, elsewhere (27), this competitive inhibitor of Cdc25 caused both G2-M however, Cpd 5 lacks significant redox activity (8). Thus, we hypoth- and G1 inhibition. esize that arylation, possibly of Cys-430 or Cys-384, is responsible for

Fig. 9. Cyclin-associated with Cdk2 or Cdk4 after Cpd 5 treatment. Synchronized tsFT210 cells were cultured for6hat32.0°C and then incubated for an additional6hinthepresence or 0 ␮M (Lane 1), 10 ␮M (Lane 2), or 20 ␮M (Lane 3) Cpd 5. The cells were harvested and sonicated in lysis buffer as described in “Materials and Methods.” A, Cdk2 immunocomplexes were analyzed by SDS-PAGE and immunoblotting with anti-cyclin A, anti-cyclin E, and anti- Cdk2. B, Cdk4 immunocomplexes were analyzed by SDS-PAGE and immunoblotting with anti-cyclin D1 and anti-Cdk4.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2000 American Association for Cancer Research. Cdc25 PHOSPHATASE INHIBITION BY VITAMIN K ANALOGUE the enzyme inhibition. This will require additional experimental studies to establish. We used the well-studied tsFT210 cell system, because the cells can be synchronized without any exogenous agents or drugs. Both Cpd 16 and Cpd 22, which are close congeners of Cpd 5, failed to block cell cycle progression. Neither Cpd 16 nor Cpd 22 had inhibitory activity against Cdc25 phosphatases in vitro. By contrast, Cpd 5 inhibited both cell cycle progression and Cdc25 phosphatase activity in vitro. These data revealed a close correlation between Cdc25 inhibition in vitro and disruption of cell cycle regulation. The observed elevated Cdc2 phosphorylation and the loss of Cdc2 kinase activity provided additional biochemical evidence for intracellular Cdc25 inhibition. Cpd 5, but not the other closely related but biochemically inactive analogues, decreased Cdc2 kinase activity in the intact cells. These concentration-response studies showing that Cpd 5 induced Cdc2 phosphorylation and inhibited its kinase activity suggest that Cpd 5 had an Fig. 11. Western blot of p53, p21, and p16. Synchronized tsFT210 cells were cultured for6hat32.0°C and then incubated for an additional6hinthepresence of 0 ␮M (Lane inhibitory effect on Cdc25B and Cdc25C within the cell and provide 1), 10 ␮M (Lane 2), 15 ␮M (Lane 3), or 20 ␮M (Lane 4) Cpd 5. Cells were also incubated a mechanistic basis for the blockage at G -M. in the presence of 0 ␮M (Lane 5), 3 ␮M (Lane 6), 10 ␮M (Lane 7), 30 ␮M (Lane 8), or 50 2 ␮ We hypothesized that inhibition of Cdc25A might mediate the G M (Lane 9) of etoposide. Cells were harvested and analyzed by Western blotting for p53, 1 p21, or p16 expression by Western blotting methods. block caused by Cpd 5, because Cdc25A seems to be important for entry into S phase (13, 31). The tyrosine phosphorylation status of both Cdk2 and Cdk4 was markedly increased by the actions of Cpd 5. intracellular inhibition of the catalytic activity of Cdc25. It is well Both of these Cdks have a central role in regulating the G1-S transition (30). Cdc25A dephosphorylates Cdk2 at Thr-14 and Tyr-15 and established that DNA damage, such as that induced by ionizing activates the functional Cdk2/cyclin E complex required for progres- radiation, produces a p53 induction and blocks the cell cycle at both sion through the S phase of the cell cycle (31). Cdc25A also controls G1 and G2-M (38). Our results indicate, however, that exposure of tsFT210 cells to Cpd 5 for 6 h did not produce p53, p21, or p16 the tyrosine phosphorylation status of Cdk4, which regulates G1 arrest by agents such as UV irradiation (37). Furthermore, the activity of induction (Fig. 11). These data suggest that the main pathway causing Cdc25A determines the phosphorylation status of Rb through its the dual cell cycle arrest by Cpd 5 is different from p53 (p21) or p16 effects on Cdk4 kinase. Our data show that Cpd 5 increased Cdk4 induction pathways. tyrosine phosphorylation, thereby decreasing kinase activity against In summary, we demonstrated that the potent K vitamin analogue, Cpd 5, inhibited an important class of growth-regulatory, dual-spec- Rb. Thus, the dephosphorylated Rb might cause the G1 block in tsFT210. ificity phosphatases and arrested cells in both G1 and G2-M phases. We cannot, however, formally exclude that Cpd 5 acts on other cell We suggest that small molecule inhibitors derived from the Cpd 5 cycle control mechanisms or on other protein phosphatases. Indeed, in pharmacophore will be useful for furthering our understanding of the hepatoma cells, Cpd 5 transiently enhanced the phosphorylation of a role of Cdc25 in regulating G1 and G2 transition and may contribute number of proteins (36, 39). Nonetheless, we found that differences to a further development of novel anticancer agents. exist in the in vitro sensitivity of several classes of protein phosphatases and that Cpd 5 induced persistent inhibition of one class of protein ACKNOWLEDGMENTS phosphatases, i.e., Cdc25. Furthermore, we have established that cell cycle arrest resulted from the cellular effects of Cpd 5 consistent with We thank Andreas Vogt, Alexander P. Ducruet, Angela Wang, and the other members of the Lazo Laboratory for their comments and scientific support. We also thank Professor Peter Wipf and members of his laboratory for synthesiz- ing SC-␣␣␦9 and Professor Michio Yamakido at Hiroshima University for his assistance. This report is dedicated to the memory of our respected colleague and friend, the late Professor Paul Dowd, who first synthesized the enzyme inhibitor naphthoquinone.

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Kenji Tamura, Eileen C. Southwick, Jeffrey Kerns, et al.

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